Role of Stress-Response Protein gp96 in the Persistence of Rheumatoid Arthritis

描述(申请人提供)：先天免疫系统和获得性免疫系统在类风湿性关节炎(RA)的发病机制中非常重要。Toll样受体对于产生获得性免疫很重要，包括T细胞和B细胞的激活。然而，TLRs在RA的持久性和破坏性中的作用尚不清楚。我们最近证明，表达突变Fas受体(LPR)的小鼠发生胶原诱导性关节炎(CIA)的频率和严重程度较低。我们发现了一种新的机制，即Fas-FasL与巨噬细胞的相互作用(M&S)在不诱导细胞凋亡的同时，通过TLR4途径增强了激活。我们发现完整的Fas-FasL信号通路促进了CIA的诱导，并增强了TLR4结扎诱导的NF-：B活化和细胞因子的表达。M&S和滑膜成纤维细胞在类风湿关节炎的发病机制中起重要作用。M&S在RA关节中同时表达Fas和FasL，并与其他M&S及滑膜成纤维细胞密切接触。这些观察表明，在RA关节中，Fas-FasL的相互作用，通过降低激活阈值，可能使滑膜M和S对TLR配体的激活敏感。我们的初步研究还表明，从RA关节分离的M和S与对照M&S相比，对微生物TLR2和TLR4配体的反应性增加，提示TLR信号可能在RA中起重要作用。我们的初步数据还表明，内质网应激反应蛋白96-kDa糖蛋白96(Gp96)在RA关节中高表达，并通过TLR2激活M和S，非凋亡性Fas-FasL相互作用促进gp96激活M&s。我们还证明了另一种潜在的TLR配体HSP22，它通过TLR4激活，在RA滑液中含量丰富。综上所述，这些观察表明，M&S之间以及M&S与滑膜成纤维细胞之间的Fas-FasL相互作用可能会使关节内局部细胞对内源性TLR配体如gp96或hsp22的激活敏感，从而导致自我持久的慢性炎症。这一应用的压倒一切的假设是，局部释放内源性TLR配体促进了关节炎从自限性到慢性疾病的进展，并且这种进展被Fas-FasL相互作用增强。具体地说，我们假设：1)利用gp96和HSP22，通过TLR结扎对RA滑膜M和S的激活将大于正常M&S或其他形式的炎症性关节炎(OIA)患者(如银屑病关节炎)患者的滑液中的激活，而RA滑膜成纤维细胞的激活将大于骨关节炎(OA)患者的滑膜成纤维细胞的激活；2)内源性TLR配体最大限度地激活TLR2或TLR4需要Fas-FasL相互作用；3)在存在但不存在完整Fas信号的情况下，gp96(或HSP22)的表达可将自限性关节炎转化为慢性进行性疾病，这依赖于TLR，以及4)gp96(和/或HSP22)的中和将改善CIA。 与公共卫生相关。类风湿性关节炎影响了至少1%的人口，而且有许多患者的治疗不充分。这项研究中提出的工作旨在确定导致类风湿性关节炎发病的新机制，这将确定新的治疗靶点。

DESCRIPTION (provided by applicant): The innate and adaptive immune systems are important in the pathogenesis of rheumatoid arthritis (RA). Toll- like receptors (TLRs) are important for the generation of adaptive immunity, including T and B cell activation. However the role of TLRs in the persistence and destruction of RA is not known. We recently demonstrated that mice expressing a mutant Fas receptor (lpr), developed less frequent and less severe collagen induced arthritis (CIA). We identified a novel mechanism whereby Fas-FasL interactions on macrophages (M&s), while not inducing apoptosis, enhanced activation through the TLR4 pathway. We demonstrated that intact Fas- FasL signaling promoted the induction of CIA and enhanced the NF-:B activation and cytokine expression induced by TLR4 ligation. M&s and synovial fibroblasts are critical in the pathogenesis of RA. M&s in the RA joint, express both Fas and FasL and are in intimate contact with other M&s and with synovial fibroblasts. These observations suggest that, in the RA joint, Fas-FasL interactions, by lowering the threshold for activation, may sensitize synovial M&s to activation by TLR ligands. Our preliminary studies also demonstrate that M&s isolated from the RA joint demonstrate increased responsiveness to microbial TLR2 and TLR4 ligands compared to control M&s, suggesting that TLR signaling might be important in RA. Our preliminary data also demonstrate that the 96-kDa glycoprotein 96 (gp96), an endoplasmic reticulum stress response protein, is highly expressed in the RA joint, and activates M&s through TLR2 and that non-apoptotic Fas-FasL interactions promote M& activation by gp96. We also demonstrate that another potential TLR ligand, HSP22, which activates through TLR4, is abundant in RA synovial fluids. Together these observations suggest that Fas-FasL interactions between M&s, and perhaps between M&s and synovial fibroblasts, may sensitize cells locally within the joint to activation by endogenous TLR ligands, such gp96 or HSP22, resulting in a self- perpetuating chronic inflammation. The overriding hypothesis of this application is that the local release of endogenous TLR ligands promotes the progression of arthritis from a self-limited to a chronic disease, and that this progression is enhanced by Fas-FasL interactions. Specifically, we hypothesize that: 1) Employing gp96 and HSP22, the activation of RA synovial M&s through TLR ligation will be greater than observed with normal M&s or those from the synovial fluid of patients with other forms of inflammatory arthritis (OIA) such as psoriatic arthritis, while the activation of RA synovial fibroblasts will be greater than observed with synovial fibroblasts from patients with osteoarthritis (OA); 2) that Fas-FasL interactions are required for maximal TLR2 or TLR4-mediated activation by endogenous TLR ligands; 3) that the expression of gp96 (or HSP22) converts self-limited arthritis to chronic progressive disease, dependent upon TLRs, in the presence, but not the absence, of intact Fas signaling and that 4) that neutralization of gp96 (and/or HSP22) will ameliorate CIA. PUBLIC HEALTH RELEVANCE. Rheumatoid Arthritis affects at least 1% of the population and there are many patients for whom therapy is inadequate. The work proposed in this study is aimed at identifying novel mechanisms contributing to the pathogenesis of rheumatoid arthritis, which would define new therapeutic targets.